3928 The Journal of Experimental Biology 212, 3928-3934 Published by The Company of Biologists 2009 doi:10.1242/jeb.034140
Thermal learning in the honeybee, Apis mellifera Tobin J. Hammer, Curtis Hata and James C. Nieh* University of California San Diego, Division of Biological Sciences, Section of Ecology, Behavior, and Evolution, Mail Code 01169500 Gilman Drive, La Jolla, CA 92093-0116, USA *Author for correspondence (
[email protected])
Accepted 1 September 2009
SUMMARY Honeybee foragers are exposed to thermal stimuli when collecting food outside and receiving food rewards inside the nest. In both contexts, there is an opportunity for foragers to associate warmth with food rewards. However, honeybee thermal learning is poorly understood. Using an associative learning paradigm (the proboscis extension reflex), we show that honeybees can learn to associate a nectar reward with a heated stimulus applied to the antenna to mimic natural contact with a warm flower or nectaroffering forager. Conditioning with longer inter-trial intervals (ITI) significantly improved learning acquisition. We also trained bees to discriminate between temperatures above (warm) and below (cold) ambient air temperature. Learning acquisition improved by 38% per 10°C increase in absolute stimulus intensity (difference between the rewarded temperature and unrewarded ambient air temperature). However, bees learned positive temperature (warm) significantly better than negative temperature (cold) differences, approximately twice as well for 10°C as compared with a –10°C difference. Thus, thermosensation, a sensory modality that is relatively unexplored in honeybees, could play a role in the acquisition of information from nestmates (social learning) and in foraging decisions influenced by associations between floral temperature and nectar rewards. Key words: honeybee, discrimination learning, classical conditioning, memory, thermoreception.
INTRODUCTION
Despite their small brain size and limited number of neurons relative to the central nervous systems of many vertebrates, social insects have evolved sophisticated learning and memory capabilities and are therefore important models for animal cognition (Dukas, 2008). In particular, honeybees have emerged as a major model system for the study of insect cognition because of their rich and intricate behavioral repertoire, complex learning and memory abilities, and the relative accessibility of their central nervous system (Giurfa, 2007). Honeybees can learn to associate floral odor, color and shape with a nectar reward and store this information in long-term memory (Hammer and Menzel, 1995). Associative learning is also important within the hive. Honeybees can identify nestmates from non-nestmates (Breed and Stiller, 1992) and can discriminate among queens or workers based on genetic similarity (Breed, 1981; Getz and Smith, 1983) using learned chemical cues. Multiple studies have examined their olfactory and visual learning, revealing a wide variety of phenomena, including learning generalization, extinction, memory spacing and lateralization (Hori et al., 2006; Letzkus et al., 2008; Letzkus et al., 2006; Sandoz and Menzel, 2001; Menzel et al., 2001; Sandoz and Pham-Delègue, 2004; Smith, 1991; Giurfa et al., 1996). However, the role of an important modality, thermosensation, in associative learning remains poorly understood, although it plays an important role in colony thermoregulation (Jones et al., 2004) and, potentially, in foraging (Stabentheiner et al., 1995). Honeybees can learn to associate thermal stimuli with a sucrose solution reward (Menzel et al., 2001) but the natural role of such learning, its characteristics and the factors regulating it remain poorly understood. In fact, insect conditioning to thermal stimuli has, to date, only been examined in depth for leaf-cutting ants, who may use their capacity for thermal learning to help locate attractive sun-exposed leaves (Kleineidam et al., 2007).
Bee foragers in the field can experience temperatures below or above ambient air temperatures when foraging for nectar inside flowers (Herrera, 1995; Kevan and Baker, 1983). In the nest, honeybee foragers returning from a good food source, such as concentrated sugar solution near the nest, can warm their bodies to higher temperatures than when returning from less concentrated or more distant sources (Stabentheiner, 2001; Stabentheiner and Hagmüller, 1991). Foragers returning from natural floral nectar and pollen sources have elevated thoracic temperatures positively correlated with colony need for these resources (Stabentheiner, 2001). Such elevated temperatures could be perceived by recruits receiving food samples (trophallaxis) from these successful foragers, because their antennae contact recruiting foragers (Tautz and Rohrseitz, 1998) and contain thermosensitive sensillae (Kovac and Schmaranzer, 1996). During trophallaxis (food exchange), nectar receivers showed proboscis temperature increases of 0.85–3.5°C (Farina and Wainselboim, 2001), increases which they should be able to perceive (Heran, 1952). Honeybee workers are therefore constantly exposed to thermal stimuli during nectar foraging and exchange. Honeybees possess paired thermoreceptive antennae (Yokohari, 1983), and thus their thermal learning may exhibit lateralization, a phenomenon observed for olfactory and visual learning (Letzkus et al., 2006; Letzkus et al., 2008). Because side-specific thermal conditioning of the PER has not been previously tested, we sought to determine if thermal learning is lateralized as well. The time period between learning trials (inter-trial interval, ITI) affects associative memory formation. Within limits, a longer ITI results in better long-term memory for the association between multiple types of sensory stimuli (including thermal) and a nectar reward (Menzel et al., 2001). We further analyzed the spacing effect on thermal learning and investigated the possibility of differential lateralization at different ITIs. We then determined how the
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Honeybee thermal conditioning magnitude of temperature differences, both positive and negative, relative to ambient air temperature, affects memory formation. We hypothesized that larger perceived temperature differences should act as more ‘salient’ thermal cues. In general, intense stimuli should more readily form associative memories (Rescorla, 1988), as demonstrated for learning discrimination and odorant concentration (Bhagavan and Smith, 1997).
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ITI experiment
To deliver the thermal stimulus, we used a custom-built probe (Fig.1A), consisting of a 12mm ⫻ 13mm ⫻ 2mm copper plate attached with thermal silver epoxy (99.8% Ag, Arctic Silver, Visalia, CA, USA) at the end of a loop of copper tubing (3.25mm diameter) through which we circulated temperature-controlled water (Haake FE2 water circulator, Thermo Haake GmbH, Karlsruhe, Germany). The ambient-air-temperature probe was a rod attached to an identical copper plate. All copper plates were half-covered with paper tape to facilitate temperature measurement with a Raytek MX6 infrared scanner (Santa Cruz, CA, USA).
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MATERIALS AND METHODS General methods
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We used the proboscis extension reflex (PER) learning paradigm to explore the ability of honeybees to associate a sucrose solution reward (the unconditioned stimulus, US) with temperature differences (the rewarded conditioned stimulus, CS+) applied to their antennae (Takeda, 1961; Bitterman et al., 1983). This technique exploits the natural response (proboscis extension) of a honeybee to nectar (US). Following the forward pairing of a CS+ with a US, the bee will extend her proboscis if she has learned to associate the sugar reward with a temperature difference. We conducted our experiments at the University of California San Diego in La Jolla, CA, USA (N32°52.690⬘, W117°14.464⬘) during January–June 2007 and January–March 2008. We randomly selected and captured honeybee foragers (Apis mellifera Linnaeus 1758) as they exited the entrances of four colonies: three colonies for the ITI experiment and one for the temperature difference experiment. We captured, chilled (4.5min at 0°C) and harnessed foragers into stainless steel tube stands (3.7cm long ⫻ 15mm wide) (Bitterman et al., 1983) (Fig.1A). Once harnessed, bees were placed in an incubator for 30min at 30°C to increase feeding motivation. Although this fasting period is relatively short compared with other studies (Bitterman et al., 1983), we found that a 30min fasting period was sufficient to achieve a strong and consistent level of PER response among experimental subjects. After fasting and before conditioning, bees were evaluated for spontaneous proboscis extension to unscented water or the control stimulus. However, response levels to these stimuli were quite low (ITI experiment: 0.8% of bees; temperature difference experiment: 2.8% of bees). After fasting, we also evaluated bees for their response to the US (sucrose solution). To do so, we touched an antenna (randomly choosing the left or right antenna) with a pipette tip with 1moll–1 unscented, analytical grade sucrose solution. Only bees exhibiting proboscis extension (approximately 80% of those tested) were used in the training procedure. All studies were conducted in a temperature-controlled room (20.3±0.7°C). Yokohari reported that honeybee antennae have thermosensitive coelocapitular sensillae that are most abundant on the most distal antennal segments (Yokohari et al., 1982, Yokohari, 1983). In all experiments, we delivered the thermal stimulus by touching only the antennal tip.
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Fig.1. (A)Schematic showing the thermal stimulus probe (TPthermal plate attached to copper tubing through which temperature-controlled water flowed) making contact with the antenna of a bee harnessed into a proboscis extension reflex (PER) stand. (B)Effect of inter-trial interval (ITI) on learning curves. Each graph shows the percentage PER response to the rewarded conditioned stimulus (CS+) and unrewarded conditioned stimulus (CS–) in each trial for the four different ITIs used in our study (data pooled for left and right treatments because no significant lateralization effect was found).
We used a forward pairing PER design. First, each bee was randomly assigned to a side-specific treatment group. We applied the thermal stimulus only to the left antenna in the left group and only to the right antenna in the right group. We trained bees to associate a thermal stimulus with a food reward over 24 trials: 12 CS+ and 12 CS– (unrewarded conditioned stimulus) trials. A CS+ trial consisted of lightly touching the designated antenna (Fig.1A) with the probe set at 31°C (10°C above ambient temperature) for 5s, followed by 2s of reward presentation (1l of 1moll–1 sucrose solution, equal to 30% sucrose w/w). Floral nectars occur at a variety of sugar concentrations (Baker and Baker, 1982), and generalist bee foragers collect nectars ranging from 10% to 70% sugar w/w (Roubik et al., 1995). To elicit proboscis extension, we first touched the designated antenna with a pipette bearing a droplet of sucrose solution and then provided the reward when the bee extended her proboscis. The CS– consisted of lightly touching the roomtemperature probe to the designated antenna for 5s without a subsequent sucrose reward. These trials were pseudorandomly
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3930 T. J. Hammer, C. Hata and J. C. Nieh
We captured, harnessed and incubated bees before conditioning as in the previous experiment. To generate temperatures above and below ambient air temperature, we used a Peltier chip (model ET1.518-F2A-H4-C1, Melcor Thermoelectric Cooler, Trenton, NJ, USA) with a metalized ceramic surface that can heat or cool to a set temperature (2–31.5°C), depending upon the voltage polarity and current applied. We attached the chip to a copper tube probe as previously described and stabilized chip temperature by circulating heated or chilled water as appropriate (Fig.1A). We built two Peltier probes, of which one served as the room-temperature probe and was not heated or cooled. We monitored probe temperatures and cleaned them as in the ITI experiment. In the ITI experiment, learning acquisition was best with an ITI of 255s and reached a plateau after 5–6 trials (Fig.2). We therefore used an ITI of 255s in the temperature difference experiment, and reduced the number of trials to 20 (10 CS+ and 10 CS– trials in the order ABBABAABABBABAABABBA). We used a 1moll–1 sucrose solution as the reward (US). Approximately 20 bees per temperature treatment were used. In some cases, we tested differences smaller than 0.25°C (Fig.3B). Thus, in order to ensure that each bee was adequately exposed to each temperature stimulus, we touched both antennae for all tested temperature differences. Left (L) bees were touched on their left antenna first, then their right antenna second. Right (R) bees were touched on their right antenna first, then their left antenna second. Each antennal contact lasted for 2.5s (total 5s exposure). In CS+ trials, the experimenter touched the pipette tip with a droplet of sucrose solution to both antennae (in the same order as the thermal stimulus) to elicit proboscis extension and then rewarded the bee. For example, L bees were touched first on their left and then on their right antenna before the reward was given. Statistical analysis
For all tests, we used JMP IN v4.04 software (SAS®, Cary, NC, USA). We analyzed learning acquisition with a discrimination index (DI), the sum of a honeybee’s responses to the CS– subtracted from the sum of her responses to the CS+ (Pelz et al., 1997). In the
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alternated over the course of the experiment in the pattern AABABBABAABABBABAABABBAB with A being a conditioned trial (CS+) and B being an unconditioned trial (CS–) (Bitterman et al., 1983). Touching the antenna provided visual and mechanical stimuli in addition to a thermal stimulus. However, the visual and mechanical stimuli were identical in CS+ and CS– trials, and thus a bee responding preferentially to the CS+ (Fig.1B) did so because she had conditioned to the thermal stimulus. We scored a bee’s response during the 5s of exposure to the CS, before the US was presented (1extension of proboscis past mandibles, 0no extension of the proboscis past mandibles). For each bee, we used one of four ITIs (30s, 105s, 180s or 255s). We chose the 30s and 180s intervals because they were used in a previous experiment testing honeybee thermal learning (Menzel et al., 2001). The remaining two ITIs were intermediate values chosen to evaluate fine-scale differences in learning performance. Thus, we chose four different ITIs [as compared with two (Menzel et al., 2001)] to better evaluate the influence of ITI on learning acquisition in our analysis model. We tested 60 bees (30 left group and 30 right group) at each ITI, for a total of 240 bees (80 bees from each colony). After trials with each bee, we thoroughly cleaned the probe plates with 100% ethanol.
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Fig.2. The effect of inter-trial interval (ITI) on thermal learning. (A) Learning curves for the total conditioned stimulus (CS+) proboscis extension reflex (PER) response over all rewarded trials are shown, divided into left (L, open circle) or right (R, filled circle) antennal treatments. The ITI corresponding to each learning curve is shown on the right, next to the results of the last trial. (B)The mean discrimination index (DI) for the different ITIs is shown, divided into left (L, open bars) and right (R, filled bars) treatments. Error bars show standard errors. Significant differences between ITI treatments (based upon Tukey HSD tests) are indicated with different letters. In this experiment, the maximum DI is 11 (the bee responds with PER to all CS+ after the first conditioning trial and no CS–) and the maximum final discrimination index (DIf) is 1.
temperature difference experiment, if a forager learns after only one trial (maximum possible learning), her DI will be 9 because she responds with PER to all CS+ after the first conditioning trial and to no CS–. To evaluate learning performance at the end of training, we also measured the ability of bees to discriminate the thermal stimulus from the control for the final stimulus trial. This index,
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Honeybee thermal conditioning
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ΔTemperature (probe temperature–room temperature) °C Fig.3. The effect of temperature (T) (cold vs warm treatments) on thermal learning. (A)Learning curves at three temperature differences (–10°C, 1°C and 10°C) over all 10 learning trials. The total conditioned stimulus (CS+) (filled diamonds) and CS– (filled squares) proboscis extension reflex (PER) responses per trial are shown (30 bees per temperature treatment). (B)Learning discrimination index (DI) values per bee (the total number of conditioned PER responses minus the total number of unconditioned PER responses per bee) are shown for the cold and warm treatments in separate plots. We also show the mean final discrimination index (DIf) value with standard error for each temperature difference, separated into cold and warm treatment plots. In this experiment, the maximum DI value is 9 (the bee responds with PER to all CS+ after the first conditioning trial and no CS–). The maximum DIf value is 1. Dashed linear regression lines with corresponding regression equations and R2 values are shown.
DIf, can be a more sensitive parameter for assaying learning and was calculated by subtracting the final CS– response from the final CS+ response. All data met assumptions of normality, and thus we
performed Student’s t-tests and analysis of variance (ANOVA). In the ITI experiment, we tested colony as a random effect (Standard Least Squares using an EMS algorithm) and ITI and lateralization
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3932 T. J. Hammer, C. Hata and J. C. Nieh (left or right treatment) as fixed effects. In the temperature difference experiments, we tested absolute temperature difference, treatment (cold or warm) and lateralization (left or right first antennal stimulation) as fixed effects. We included all appropriate fixed-effect interactions in our full models. We used t-tests to evaluate the hypothesis that bees can discriminate between the rewarded thermal stimulus and the unrewarded control stimulus (mean DI is greater than zero). We performed paired t-tests to determine if responses to CS+ were significantly greater than responses to CS– for the same individual. For comparisons of variance, we used Levene’s test for equality of variances. Where appropriate, we applied a Sequential Bonferroni correction using the Dunn–Sidak method to correct for type I error (Sokal and Rohlf, 1995) and note if a result is significant (*) or not (NS) after this correction. Pairwise comparisons were performed with post-hoc Tukey–Kramer Honestly Significant Difference (HSD) tests. RESULTS ITI experiment
Honeybees learned to associate a temperature difference with a food reward, and learning acquisition increased with reinforcement (Fig.1B, Fig.2). Out of 240 honeybees used, over 70% displayed at least one response to the CS+ after conditioning trials, and responses increased over the course of training (Fig.1B). The DI and DIf indices were significantly greater than zero for all ITI greater than 30s (Table1). For DI, but not DIf, the 30s right antennal treatment group also showed significant learning (Table1). Thus, bees successfully distinguished between the heated probe (CS+) and the room-temperature probe (CS–) for ITI ≥30 s. Analysis of DI (learning summed over all trials) revealed no significant interaction between ITI and lateralization (F1,2351.2, P0.28), and thus we ran a simplified three-factor model. In this model, there was no significant effect of colony (F1,2362.4, P0.12) or lateralization (F1,2360.0004, P0.98). However, there was a highly significant effect of ITI (F1,23642.0, P
